COVID-2019 Genome Sequence Analysis: Phylogenetic Molecular Evolution and Docking of Structural Modelling of Receptor Binding Domain of S Protein in Active Site of ACE2

Meanwhile the outbreak of the Covid-19 since December, 2019 in China, it has killed more than a hundred thousand of people of all ages and sex across the globe in a short span of time. On the bases of this study the nearest family member of the virus and its receptor binding domain of S protein including its model structure and function of its active sites were naked through Multiple Sequence Alignment, modelling and molecular docking software accordingly its repository genome databases. The virus was genetically associated and molecular evolutionary related with (RaTG13) and it scores 96.12% homology with 99% query coverage followed by bat-SL-CoVZC45 and bat-SL-CoVZXC21 notch 89.12% and 88.65% respectively. However, SARS and MERS corona type virus those outbreak earlier respectively less likely family members of 2019-nCoV. Though the virus has a close genetic association with those previous SARS coronaviruses, and certainly the spike protein used as a binding receptor to fight against human receptor protein of ACE 2, but on the basis of FRODOC and HDOCK server analysis multi favorable active sites of S protein was discovered such GLN493 shown as a finest key in both model and possessed a unique traits on it resulting unexpected rate of transmission and number of people died while compared to the previous one. TYR500, ASN501, GLN498 and others residues preferably contemplate site also. In particular, the diversity of the virus in the world may be due to the genome structure of the virus and S gene changed over the time, across the world against to host of human genetic diversity, which may be more robust, and may be a new and unique feature. This is because it is characterized close to contact with distance divergence between wild type novel coronavirus which was risen from China against to the genomes from Lebanon, India, Italy, and USA and so on. Thus, the World Health Organization and its researchers should focus on immunologic research and effective drug and vaccine development that will help to address the epidemiology of the virus, which can provide a long-term solution.

The angiotension converting enzyme (ACE) gene I/D polymorphism in different ethnic groups of geriatric age living in the Far North

The study of ACE gene I/D polymorphism has been carried out in elderly, senile and long-liver patients with coronary heart disease (CHD) taking into account their nationality, age and sex. It has been recorded that with the increase of age there is a decrease in the frequency of the genotype ACE*I/*I and a tendency of increase in the frequency of the genotype ACE*D/*D. A comparative analysis of genotypes АСЕ*D/*D and АСЕ*D/*I has showed sex differences in the frequency of homozygous genotype detection. Left ventricular hypertrophy can be observed significantly more often among carriers of genotype ACE*I/*I established by Sokolow-Lyon ECG signs. Association analysis of ACE gene I/D polymorphism has registered significant differences in BMI and blood lipid parameters.

Association of ACE Gene with Metabolic Syndrome, Hypertension and Alzheimer Disease by RFLP-PCR

ACE gene is associated with multifactorial diseases:metabolic syndrome,obesity,diabetes mellitus,hypertension,stroke,myocardial infarction,ageing,Alzheimer disease,acute pulmonary failure and COVID-19 infections.The purpose of this study is to test the association between the II,ID and DD polymorphisms of angiotensin-converting enzyme(ACE)gene,and metabolic syndrome,hypertension(HBP)and Alzheimer disease(AD),based on clinical data and biochemical laboratory investigations conducted on inpatients,applying the RFLP-PCR technique.The genotyping of ACE gene was carried out by RFLP-PCR on the basis of DNA isolated from total blood in 144 subjects selected at Giurgiu County Emergency Hospital.The results were statistically processed by the Hardy-Weinberg equilibrium,Odds Ratio,VMD,StatsDirect and PyElph software.The II,ID and DD polymorphisms of ACE gene identified by the RFLP-PCR present a high risk of developing the metabolic syndrome in the MS,hypertension and Alzheimer disease groups.

Construction of Vector for Functional Analysis of the Intrinsically Bent DNA in the ACE3 Replicator from <i>Drosophila melanogaster</i>

DNA replication is a crucial process for species survival, nevertheless it is not clear which factors define origin selection in multicellular eukaryotes. Developmental gene amplification systems, such as the one described during ovarian follicles development in Drosophila melanogaster, are useful tools for studying of DNA replication process in these organisms. We previously described that the well characterized third chromosome amplified domain of D. melanogater displays three intrinsically bent DNA sites: b1, localized at an amplification control element (ACE3), b2 and b3, both localized at the preferential origin ori-β. This proposal aimed to construct a Drosophila transformation vector, which contains a short deletion at the ACE3, in order to reduce the intrinsically bent DNA site b1, and analyze the functional role of this site in the gene amplification process. Through a series of cloning steps, we obtained a Big Parent vector derivative, containing a deletion at the positions 176-180 bp, inside the ACE3. The generation of a Drosophila transformation vector displays a reduced intrinsically bent DNA site in the third chromosome amplified domain, it will allow the analysis of the functional role of this curvature in developmental gene amplification, providing new insights on replication initiation in D. melanogaster and the function of intrinsically bent DNA sites.

河北地区汉族人非胰岛素依赖型糖尿病眼底病变与ACE基因多态性的相关性研究

目的 探讨血管紧张素Ⅰ转换酶（ＡＣＥ） 基因插入／ 缺失（Ｉ／Ｄ） 多态性与非胰岛素依赖型糖尿病（ＮＩＤＤＭ） 及并发眼底病变的关系。方法 以ＡＣＥ基因１６ 内含子的１ 个２８７ｂｐ 的Ａｌｕ 序列为多态性标志，用ＰＣＲ方法扩增基因片段，６ ％ 非变性聚丙烯酰胺凝胶电泳检测ＰＣＲ 产物。结果１４９ 例ＮＩＤＤＭ 与１００ 例正常人基因型频率与等位基因频率差异无显著性。ＤＤ 型与Ｄ 等位基因在ＤＲ（ ＋ ） 组出现频率高，ＤＲ（ ＋ ） 组与ＤＲ（ － ） 组相比，基因型及等位基因分布差异具显著性。结论 ＡＣＥ 基因多态性与ＮＩＤＤＭ

小桐子早期光诱导蛋白基因ELIP克隆及其与靶向miRNA的互作与低温下共表达分析

【目的】作为一种喜温冷敏植物,低温严重影响小桐子的生长发育、地域分布与产量。前期工作发现12℃低温锻炼可显著提高小桐子的抗冷性,小桐子早期光诱导蛋白(ELIP)基因是低温高响应基因。为探究JcELIP在小桐子低温响应中的作用,全面了解JcELIP的结构、调控机制、进化关系及JcELIP与miRNAs的互作关系,也为后续小桐子抗冷分子育种提供一个重要的候选基因资源。【方法】通过RT-PCR克隆了小桐子JcELIP基因并对其进行系统的生物信息学分析,采用RT-qPCR分析JcELIP基因在根、茎、叶中及12℃低温锻炼过程中的表达变化,鉴定与JcELIP互作的miRNAs,进行了12℃低温锻炼过程中的共表达分析。【结果】该基因完整的开放阅读框(ORF)为585 bp,编码194个氨基酸,蛋白的分子质量为2.04 kD,理论等电点为9.59,属于稳定的疏水碱性蛋白,具有3个疏水跨膜螺旋;三级结构主要由ɑ-螺旋和无规则卷曲组成,具有叶绿素a/b结合位点。顺式作用元件预测显示JcELIP具有脱落酸等激素响应元件。进化分析显示:小桐子JcELIP与木薯MeELIP同源性最高。RTqPCR分析显示:正常生长条件下小桐子JcELIP在根、茎、叶中的表达量无显著差异;12℃冷锻炼条件下JcELIP在叶中表达快速上调,48 h时其表达量达到对照组的64.8倍,表明JcELIP参与了小桐子对冷胁迫的响应与适应。基于小桐子降解组数据,鉴定到miR390-x、miR6476-x和novel-m0090-3p等8个miRNAs对JcELIP的表达具有调控作用;共表达分析结果表明在12℃低温锻炼过程中JcELIP的表达受miR390-x和novel-m0090-3p显著负调控。【结论】JcELIP高响应低温胁迫并与miRNAs互作,表明其在小桐子响应低温胁迫过程中发挥重要作用。

TMPRSS2 Polymorphism and Its Correlation with SARS-CoV-2 Susceptibility - A Secondary Publication

Background:Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus affected more people than SARS-CoV-1 and related coronaviruses.Human genetic factors,besides respiratory droplets and direct exposure to the virus,are found highly responsible for transmitting SARS-CoV-2 infection.Aim:The objective of this study was to determine the plasma levels of the TMPRSS2 gene and its role in SARS-CoV-2 susceptibility.Methodology:A total of 100 patients,i.e.50 SARS-CoV-2 positive patients as the case group and 50 SARS-CoV-2 negative samples as the control group,were selected randomly and included in this case-control study to determine the association between TMPRSS2 gene and susceptibility to SARS-CoV-2 infection and severity of coronavirus disease 2019(COVID-19).The TMPRSS2 levels of case and control samples were measured through an enzyme-linked immunosorbent assay(ELISA).Following the genomic DNA extraction,a set of reverse and forward primers of human TMPRSS2 gene primers were used for the amplification of the TMPRSS2 gene.Results:In the control group,the ratio of men to women was more or less the same while in the case group 62%of the population were women.The TMPRSS2 level was found to be 4.70±7.7 ng/ml in case samples while it was 4.73±5.7 ng/ml in control samples.Conclusion:The levels of TMPRSS2 in plasma samples of both controls and cases were found to be the same indicating that the entry of SARS-CoV-2 is not dependent on the plasma levels of this protein.

Correlation of angiotensin converting enzyme gene polymorphism with perioperative myocardial protection under extracorporeal circulation

Objective:To observe the expression of angiotensin converting enzyme(ACE),angiotensinⅡ(AngⅡ),cardiac troponin 【cTnⅠ),creatine kinase isozymes(CK-MB) and muscle red protein(Myo) after cardiopulmonary bypass(CPB),and to investigate the association of polymorphisms in angiotensin converting enzyme genes and myocardial injury.Methods:Sixty-three patients suffered from rheumatic mitral stenosis and scheduled for mitral valve replacement with CPB, were randomly divided into three groups according polymorphisms in angiotensin converting enzyme genes:typeⅡ,type ID,type DD(each=21).Blood samples were withdrawn from artery before operation(T1),at the beginning of CPB(T2),30 min after CPB(T3),(T4) at the end of CPB(T5), 2 h after CPB(T6),6 h after CPB(17) to measure the expression of ACE,AngⅡ,cTnⅠ,CK-MB, Myo.Results:The level of ACE during and after CPB were significantly higher than those before CPB(P【0.05).As extension of CPB time,the expression of ACE was increased.The level of cTnⅠ, CK-MB,Myo after CPB were significantly higher than those before CPB(P【0.05).The level of cTnⅠ,CK-MB and Myo were highest at T7,T6 and T5 and T7,respectively.The level of ACE,AngⅡ,cTnⅠin patients with DD genotype was significantly higher than the ID andⅡgenotype(P【 0.05).Besides,the level of ACE,AngⅡin patients with ID genotype was significantly higher than the II(P【 0.05).Conclusions:There is certain correlation between CPB perioperative midterm ACE and cTnⅠ,Myo,CK-MB.ACE DD genotype is a susceptibility gene of the CPB perioperative myocardial injury.

Interaction mechanism of egg white-derived ACE inhibitory peptide TNGIIR with ACE and its effect on the expression of ACE and AT1 receptor

The egg white-derived hexapeptide TNGIIR inhibits angiotensin-converting enzyme(ACE)activity in vitro.In this work,molecular docking revealed that TNGIIR established hydrogen bonds with the S1(Ala 354),S2(Gln 281,His 513,Tyr 520 and Lys 511)and S1(Glu 162)pockets of ACE.In addition,the potential antihypertensive effect of the oral administration of TNGIIR in spontaneously hypertensive rats(SHR)was investigated,as was the effect of this peptide on the mRNA expression of ACE and angiotensin type 1(AT1)and type 2(AT2)receptors in renal tissue.The oral administration of TNGIIR(2,10 and 50 mg/kg)for up to four weeks did not reduce the blood pressure of SHR,in contrast to captopril(10 mg/kg,orally),but attenuated the mRNA expression of ACE and AT1 receptor(as did captopril).In contrast,both TNGIIR and captopril enhanced the expression of AT2 receptor mRNA.There was no change in the circulating concentration of angiotensin I,but a slight decrease(about 10%)was seen in the concentration of circulating angiotensin II with TNGIIR and captopril.

Study of Angiotensin Converting Enzyme Gene Polymorphism in Egyptian Type 2 Diabetes Mellitus with Diabetic Kidney Disease

Objective: Diabetic kidney disease DKD (Diabetic nephropathy DN) is considered one of the chronic micro vascular complications of diabetes mellitus and considered the commonest cause leading to chronic renal failure and chronic renal dialysis. Genetic susceptibility has been implicated in DKD. The angiotensin converting enzyme (ACE) is one of the key roles in the renin angiotensin system cascade by converting angiotensin I to angiotensin II which plays a key role in regulation of blood pressure as well as electrolytes and fluid balance. This study addressed the association of (ACE) gene polymorphisms with DN in Egyptian (T2DM) patients. Methods: Our research comprised of 75 cases of T2DM with diabetic kidney disease, 100 cases of T2DM without DKD and 94 healthy volunteers. Different genotypes of ACE gene were determined by SSP-PCR analysis. Results: Gene polymorphism of ACE (DD, ID, II) in diabetic patient with DKD is 44%, 52%, 4% respectively and for T2DM individuals without DKD is 23%, 72%, 5% respectively. (DD) had significant higher frequencies in T2DM patients with DKD compared to those without DKD (p < 0.005) and (ID) had significant higher frequencies in T2DM without DKD (p < 0.0001). These results indicated that there is an association between ACE gene polymorphisms and susceptibility of diabetic patients to be affected by diabetic kidney disease. Conclusion: From our results, we can conclude that genotype of ACE in Egypt DD is the genotype of cases diabetic kidney disease. So the presence of D allele has a significant relation with diabetic kidney disease. Our data confirm the role of ACE in its relationship with diabetic kidney disease in Egyptian type 2 diabetic patients.

基于生物信息学分析乌司奴单抗治疗寻常型银屑病不应答的关键基因和信号通路

目的应用生物信息学技术寻找乌司奴单抗治疗寻常型银屑病无应答患者中的差异表达基因,分析信号通路,探讨不应答机制。方法在Gene Expression Omnibus(GEO)下载GSE117239数据库。以P<0.05及|logFC|≥1.5为标准筛选表达差异基因。通过String数据库进行蛋白互作网络(PPI)分析,使用Cytoscape的cytohubba插件提取关键目标和子网络。使用Funrich对表达差异基因进行GO分析,最后使用ConsensusPathDB数据库进行KEGG pathway分析。结果发现76个差异基因,其中上调基因48个,下调基因28个。GO分析显示上调基因主要富集的生物过程为细胞定位、免疫效应过程、细胞组分生物合成、细胞活化、细胞死亡。下调基因主要富集的生物过程为调节血液循环、血管生成、血管发育以及组织迁移等。KEGG分析显示差异基因主要富集的信号通路为:核糖体、凋亡、多糖降解、溶酶体等信号通路。PPI网络分析出的10个枢纽基因为:CCL20、CXCL9、S100A9、S100A12、ARG1、OAS1、OASL、IL36G、SERPINB4、KRT16。结论CCL20、CXCL9、S100A9等基因可能在乌司奴单抗治疗寻常型银屑病不应答中发挥关键作用。

基于基因测序技术分析肛瘘患者微小RNA的表达特点及功能

目的采用基因测序技术,检测肛瘘瘘管壁组织中异常表达的miRNA,并进一步分析相关信号通路的功能。方法选择高位复杂性肛瘘患者3例均行手术治疗,取各患者手术切除的瘘管壁组织及瘘管壁远端正常组织形成自身配对对比,采用高通量测序技术筛选瘘管壁组织中差异表达的miRNA并进行聚类分析。采用TargetScan、miRanda两个靶基因数据库,对差异表达的miRNA进行靶基因预测。最后再选取两个数据库交叉的靶基因进行GO和KEGG富集分析,预测靶基因参与的生物学过程及信号通路。结果本研究共计筛选出20个表达差异具有统计学意义的miRNA。通过靶基因预测,共计得到85510个靶基因。进一步行GO富集分析结果提示:本研究得到的差异表达的20个miRNA靶基因所涉及的参与的生物过程主要包括:转移酶活性、调控转录、三磷酸腺苷结合、金属离子结合、DNA结合、核酸结合、核苷酸结合、细胞骨架、细胞投影、细胞连接、RNA聚合酶对转录的正调控作用、胞内信号转导等。通过KEGG富集分析显示,与本研究中差异表达的20个miRNA密切相关的信号通路主要包括癌通路、Ras、Rap1、PI3K-Akt、cAMP、MAPK、Hippo等通路。结论肛瘘瘘管壁组织中特征性的miRNA主要参与细胞增殖、迁移、凋亡相关的信号通路,与创面愈合密切相关,这些通路可能成为临床促进肛瘘术后创面愈合的潜在治疗靶点。

257例肛门及肛管尖锐湿疣组织HPV感染的基因分析

目的探讨肛门及肛管尖锐湿疣病变中人乳头瘤病毒(human papillomavirus,HPV)感染的基因型别及临床意义。方法从257例肛门及肛管尖锐湿疣(condyloma acuminata,CA)石蜡组织标本中提取23种HPV DNA,采用基因扩增结合基因芯片技术对其组织的23种HPV基因型别进行检测,并对其患者进行临床病理资料分析。结果 257例肛门及肛管CA病变组织中检出HPV阳性者183例,HPV感染率为71.21%(183/257)。其中一重HPV感染139例,阳性检出率为54.09%(139/257),多重HPV感染44例,阳性检出率为17.12%(44/257)。一重HPV感染中HPV6型66例,阳性检出率为25.68%(66/257),HPV6型是最主要的感染型别;其次是HPV11型,共有64例,其阳性检出率为24.90%(64/257)。多重HPV感染中,HPV6+11型23例,占多重感染的52.27%(23/44),是多重感染的主要型别;其次是HPV6+11+16型3例,占多重感染的6.82%(3/44)。结论 HPV6型、11型、6+11型、6+11+16型感染是肛门及肛管CA的主要致病类型。基因扩增结合基因芯片技术是一种比较适合临床进行高、低危HPV分型检测的诊断方法,其敏感性高、特异性好。

肛管及肛门区尖锐湿疣组织中人乳头瘤病毒基因类型的研究

目的高危型人乳头瘤病毒(high-risk human papillomavirus,HPV)感染是宫颈癌的主要致病原因,现已发现HPV感染可诱发人类的许多肿瘤和疣。文中旨在探讨南京地区肛管及肛门区尖锐湿疣(condyloma acuminata,CA)患者中HPV基因类型的感染情况及其临床意义。方法从86份肛管及肛门区CA石蜡组织标本中提取23种HPVDNA,采用基因扩增芯片技术对其组织进行23种HPV基因类型的检测,并对其患者进行临床病理资料分析。结果 86份肛管及肛门区CA患者组织标本中检出HPV阳性66例,感染率为76.74%(66/86),其中单一型的阳性检出率为56.98%(49/86);单一型的感染中HPV11型为26例,其阳性检出率为30.23%(26/86),是最主要的感染类型;其次HPV6型为23例,阳性检出率为26.74%(23/86);混合型HPV感染17例,阳性检出率为19.77%(17/86);其中HPV6+11型8例,占混合型感染的47.06%(8/17),是混合型感染的主要类型;其次是5例4种类型的混合感染,占混合型感染的29.41%(5/17)。结论 HPV11型、6型、6+11型、4种类型的混合感染是肛管及肛门区CA的主要致病类型,基因扩增芯片检测技术是一种比较适合临床进行HPV分型检测的、敏感性高和特异性好的诊断方法,尤其适合开展某种病变中HPV感染的分子流行病学的研究。

胃癌c-erbB-2过度表达与预后的关系

探讨c－erbB－2过度表达与胃癌预后的关系。方法：用免疫组化ABC法对103例胃癌手术标本及151个转移淋巴结进行c－erbB－2表达检测。结果：21．4％胃癌手术标本出现阳性表达．其中进展期胃癌、乳头状腺癌、高中分化胃癌及伴淋巴与肝转移的胃癌阳性率显著增高（P＜0．05与＜0．01）；转移淋巴结表达阳性率高于胃癌原发灶（X2＝3．7．P＞0．05）。高中分化胃癌伴c－erbB－2过度表达者5年生存率显著低于阴性者（P＜0．01）。结论：c－erbB－2过度表达可作为胃癌预后估计指标之一。

针刺“风府”穴对大鼠脑内神经肽胆囊收缩素基因表达的影响

手法针刺大鼠“风府”（天门）穴，用ＮｏｒｔｈｅｒｎＢｌｏｔ方法对针刺不同时限的生理状态大鼠脑内神经肽胆囊收缩素（ＣｈｏｌｅｃｙｓｔｏｋｉｎｉｎＣＣＫ）的基因在转录水平的表达情况进行比较研究。结果表明针刺即刻ＣＣＫ信息核糖核酸（ｍＲＮＡ）表达开始增加，３小时其浓度急剧升高．６小时已回落，２４小时几近平复。提示针刺后３～６小时是ＣＣＫ基因表达的高峰时期，手法针刺诱发的脑内ＣＣＫｍＲＮＡ的表达没有观察到累积迭加和持续促进的结果，并且与捉抓引起的应激反应有本质区别。

女性肛门及肛管尖锐湿疣组织HPV感染基因型谱的分析

目的探讨女性肛门及肛管尖锐湿疣组织中人乳头瘤病毒(HPV)感染的基因型别及其临床意义。方法从140例女性肛门及肛管尖锐湿疣(CA)石蜡组织标本中提取23种HPV-DNA,采用基因扩增结合基因芯片技术对其组织进行23种HPV基因型别的检测,并对其患者进行临床病理资料分析。结果 140例女性肛门及肛管CA组织标本中检出HPV阳性者103例,总的HPV感染率73.57%(103/140)。其中一型HPV感染68例,阳性检出率为48.57%(68/140),多型HPV感染35例,阳性检出率为25.00%(35/140)。一型HPV感染中HPV11型为34例,阳性检出率为24.29%(34/140),HPV11型是最主要的感染型别;其次是HPV6型,共有27例,其阳性检出率为19.29%(27/140)。多型HPV感染中,HPV6+11型13例,占多型感染的37.14%(13/35),是多型感染的主要型别;其次是HPV11+18型和HPV6+11+16型各3例,各占多型感染的8.57%(3/35)。结论 HPV6型、11型、6+11型、11+18型和6+11+16型感染是女性肛门及肛管CA的主要致病类型。基因扩增结合基因芯片技术是一种比较适合临床开展HPV分型检测的诊断方法,其敏感性高,特异性好。尤其适合HPV感染的分子流行病学的研究。

质粒介导的RNA干扰对AD293细胞中HBcAg表达的抑制作用

目的研究质粒介导的RNA干扰效应对HBcAg基因表达的抑制作用。方法设计并构建针对HBcAg基因的小发夹RNA表达载体,将构建好的shRNA表达载体和HBcAg-增强型绿荧光蛋白融合蛋白表达载体共转染人胚肾细胞株AD293,以空载体组以及针对无关序列的shRNA表达载体组为阴性对照,流式细胞术和实时荧光定量PCR法检测RNAi的抑制效果。结果构建的特异性shRNA表达载体可以抑制HBcAg基因在AD293细胞中的表达,流式细胞仪检测抑制率可达76%,实时荧光定量PCR法检测HBcAg基因的mRNA,抑制率可达58.6%。结论质粒介导的RNAi可以有效抑制HBcAg基因的表达,为利用RNAi进行抗乙型肝炎病毒复制研究提供了一个可供选择的方法。

一个可能的与人端粒结合因子相互作用蛋白Tara的分离和克隆

目的 :分离和鉴定端粒结合因子 (TRF1)免疫共沉淀蛋白复合物并克隆其基因。方法 :以 TRF1抗体应用免疫共沉淀方法 ,从细胞蛋白抽提物中分离 TRF1蛋白复合物 ,并作蛋白质肽指纹谱鉴定 ;应用温度梯度 PCR,从人 c DNA文库中扩增目的基因 ,并构建 p EGFP- C2真核表达载体 ;核苷酸测序和 Western blot验证目的基因序列和表达载体。结果 :从 TRF1免疫沉淀复合物中分离出了 Tara蛋白 ;人 c DNA文库中 PCR扩增所得产物为 1.7kb,与 Tara基因 CDS序列同源性为 99.9% ;GFP/ Tara融合蛋白约 10 0 k D,Tara在间期 He La细胞中弥散表达于胞浆 ,而有丝分裂细胞则定位于整个细胞。结论 :Tara是一个可能的 TRF1相互作用蛋白 ,其作用机制需进一步实验研究。

问号钩端螺旋体lipL32/1-lipL21-OmpL1/2融合基因原核表达及其产物免疫原性分析

目的:构建问号钩端螺旋体(简称钩体)lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统,优化目的产物表达条件并对表达产物免疫原性进行鉴定。方法:采用连接引物PCR构建lipL32/1-lipL21-OmpL1/2融合基因,并用常规基因工程方法建立其原核表达系统。采用SDS-PAGE及Bio-Rad凝胶图象分析系统,检测目的重组蛋白rLipL32/1-LipL21-OmpL1/2表达量。采用免疫双扩散试验及WesternBlot,鉴定rLipL32/1-LipL21-OmpL1/2的免疫原性。结果:获得了序列正确的lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统E.coliBL21DE-3pET42a-lipL32/1-lipL21-ompL1/2。表达条件优化后的rLipL32/1-LipL21-OmpL1/2产量为37.78mg/L,是优化前的3.7倍。rLipL32/1-LipL21-OmpL1/2兔抗血清免疫双扩效价为1∶4。rLipL32/1-LipL21-OmpL1/2抗血清能识别rLipL32/1-LipL21-OmpL1/2以及rLipL32/1、rLipL21、rOmpL1/2。rLipL32/1-LipL21-OmpL1/2能与问号钩体56601株全菌兔抗血清以及黄疸出血群、流感伤寒群、波摩那群、秋季群问号钩体感染患者血清出现阳性杂交信号。结论:成功地构建了lipL32/1-lipL21-OmpL1/2融合基因及其原核表达系统,表达产物具有良好的抗原性和交叉免疫反应性,可作为研制通用型问号钩体基因工程疫苗及通用型钩体病血清学检测的抗原。